Functional and Biochemical Analysis of the N-terminal Domain of Phytochrome A

Abstract

Phytochrome A (phyA) is a versatile plant photoreceptor that mediates responses to brief light exposures (very low fluence responses, VLFR) as well as to prolonged irradiation (high irradiance responses, HIR). We identified the phyA-303 mutant allele of Arabidopsis thaliana bearing an R384K substitution in the GAF subdomain of the N-terminal half of phyA. phyA-303 showed reduced phyA spectral activity, almost normal VLFR, and severely impaired HIR. Recombinant N-terminal half oat of PHYA bearing the phyA-303 mutation showed poor incorporation of chromophore in vitro, despite the predicted relatively long distance (>13 Å ) between the mutation and the closest ring of the chromophore. Fusion proteins bearing the N-terminal domain of oat phyA, ␤-glucuronidase, green fluorescent protein, and a nuclear localization signal showed physiological activity in darkness and mediated VLFR but not HIR. At equal protein levels, the phyA-303 mutation caused slightly less activity than the fusions containing the wild-type sequence. Taken together, these studies highlight the role of the N-terminal domain of phyA in signaling and of distant residues of the GAF subdomain in the regulation of phytochrome bilin-lyase activity.Plant photoreceptors monitor the cues provided by the light environment and trigger modifications that adjust growth and development to the prevailing conditions (1, 2). Phytochromes are sensors of red and far-red (FR) 3 light that bind an open chain tetrapyrrole chromophore (3, 4). Arabidopsis thaliana bears five phytochrome apoprotein genes (PHYA through PHYE) (5). phyA is a versatile photoreceptor that induces seed germination in response to brief exposures to light (6, 7), which the seeds may experience during soil labor. phyA is also required to perceive the prolonged exposures to FR that the seedlings experience when they emerge from the soil under dense plant canopies (8). These two photoresponses mediated by phyA are of the types called very low fluence responses (VLFR) and high irradiance responses (HIR), respectively (9). Some physiological processes (e.g. inhibition of hypocotyl growth, unfolding of the cotyledons) exhibit both VLFR and HIR as two discrete phases of response, where VLFR saturates with infrequent FR pulses and HIR requires very frequent or continuous FR (10). HIR requires cis-acting elements at target gene promoters (11) and domains of the phyA molecule itself (12) that are dispensable for VLFR.The N-terminal domain of phytochromes bears the chromophore attachment site (13) and provides differential spectral selectivity to phyA (more active under FR than red light) compared with phyB (more active under red light than FR) (14). The C-terminal domain contains a histidine kinase-related sequence motif that is able to mediate phosphorylation (15, 16), two PAS (domain named after Per, Arnt, and Sim) motifs important for downstream signaling in the context of the full molecule (17), and residues necessary for dimerization (18,19) and targeting to the nucleus upon light activation (20, 21)...

Lysine 206 inArabidopsisphytochrome A is the major site for ubiquitin-dependent protein degradation

“…Some point mutations in the N-terminal domain of phyA adversely affect the biological functions of phyA (43). However, most point mutations of phyA that involved substitution of conserved lysines by arginines in the N-terminal domain had normal phyA activity, with the exception of K202R.…”

Evolutionary Studies Illuminate the Structural-Functional Model of Plant Phytochromes

“…The N-terminal extension ( Figure 3B) is unique to phytochromes in embryophytes and their algal relatives; it is present in the phytochromes of zygnemetalean green alga, which diverge from the tree before Coleochaetales and Charales (the closest algal relatives of embryophytes; Delwiche et al, 2004) but is not present in prokaryotic phytochromes, and no crystal structure for this region is available. It is a region that in PHYA may harbor sites important for protein stability and spectral integrity and in PHYB that are important for efficient signaling (Quail, 1997;Mateos et al, 2006;Trupkin et al, 2006;Kneissl et al, 2008;Oka et al, 2008). The C-terminal regions of plant PHY and of those prokaryotic PHY that have been sequenced are not homologous (Lamparter, 2004;Mathews, 2006); in green plants, this region comprises two PAS and single His kinase and ATPase domains ( Figure 3B), the latter two domains form the core of the His kinase-related domain (HKRD; Rockwell et al, 2006).…”

Light-Independent Phytochrome Signaling Mediated by Dominant GAF Domain Tyrosine Mutants ofArabidopsisPhytochromes in Transgenic Plants

“…Although the majority of phytochrome mutants so far identified have been loss-of-function alleles (for a recent list of phy mutant alleles, see Supplemental Table 2 in ), alleles of phytochromes with increased sensitivity to light and weak constitutive photomorphogenesis (COP) phenotypes have also been reported (Kretsch et al, 2000;Weller et al, 2004;Dieterle et al, 2005;Mateos et al, 2006). The latter are still dependent on light for function, so their functions cannot be analyzed without activating other photobiological processes.…”